The present invention relates to a method for extracting lipids from tissue samples for the purpose of storing and preserving the tissue samples and the product of that method. The method of the present invention is used to particular advantage as a storage solution prefatory to the cross-linking of the intact collagen fibrils of collagenous tissue samples. In more detail, the present invention relates to a high osmotic pressure (compared to physiologic) solution which has particular advantage when used as a storage medium for samples of collagenous tissue before cross-linking the collagen in the tissue with any number of methods including glutaraldehyde and/or in accordance with the method described in the above-referenced U.S. Pat. No. 5,147,514.
Photooxidative cross-linking of the collagen fibrils of tissue samples such as bovine pericardium in accordance with the method of U.S. Pat. No. 5,147,514 results in a product having physical and chemical properties which make that product particularly suitable for use as a biomaterial for use as an artificial tendon, heart valve, or pericardial patch. Such biomaterials are characterized by several properties which confer upon them significant advantages over previously available materials used as bioprosthetics. They are produced by harvesting a sample of such tissue, incubating the sample in an aqueous media solution of a photooxidative catalyst buffered to about physiological pH (6.8-8.6), and then irradiating with light to cross-link the collagen.
It was discovered that it was advantageous to xe2x80x9cpreconditionxe2x80x9d the tissue sample by incubation in a media solution which did not include the catalyst before transfer to the solution including the catalyst for irradiation. When preconditioned in this manner, the resulting product shows decreased susceptibility to proteolytic degradation. It was also discovered this high osmolality, first media solution not only gives desirable results when used to precondition the tissue sample before cross-linking by that photooxidative process but also when used to store the tissue sample before cross-linking with glutaraldehyde and other processes as known in the art. The method and product of the present invention appear to achieve this result by removing, or decreasing the content, of the non-collagenous components of the tissue sample, including, in particular, extracting lipids from the tissue sample. Because lipids, and particularly phospholipids, are the main component of the membranes of living cells, it appears that extraction of lipids has the desirable effect of devitalizing the tissue sample, thereby reducing the bioburden of the tissue sample. A reduction in bioburden is a strong indicator of a longer shelf life of the tissue sample, and for this reason, the high osmolality solution of the present invention is advantageously used as a storage medium for the collagenous tissue sample.
It has long been standard practice, for instance, in histological laboratories, to store tissue samples in alcohol at low temperature and to use freeze drying if the sample is to be preserved for longer periods of time. Standard practice for storage of samples for relatively short periods of time usually involves incubation in one of several known saline solutions, buffered to physiological pH, at low temperature. However, both methods jeopardize the maintenance of the native fibrillar structure of structural proteins such as collagen.
The maintenance of collagen fibrillar structure is of particular concern in light of experimental data indicating that the method described in U.S. Pat. No. 5,147,514 results in the cross-linking of the collagen fibrils in their true, native state, e.g., as intact collagen fibrils, and that this capability of that method appears to be responsible for the excellent mechanical properties of the resulting product and the ability of the product to resist in vivo degradation. It is, therefore, an object of the present invention to provide a process for extracting lipids from collagenous tissue samples before photooxidative cross-linking of the collagenous tissue sample. However, it is apparent from the results obtained when used in that process that the media solution and lipid extraction process of the present invention are useful in preserving tissues for other cross-linking processes, such as acyl azide, polyglycidylethers, carbodiimide, and glutaraldehyde cross-linking, and other cross-liking processes known in the art, for preservation of any proteinaceous material or tissue, and perhaps even more broadly, as a storage medium for many different types of tissues, biomaterials, and/or extracts or solutions of same and/or their component parts or molecules.
In a broad sense, therefore, it is an object of the present invention to provide a method for preserving biological specimens, tissues, extracts, biomolecules, and/or isolates which both maintains the native state of the sample and helps protect the sample from damage or degradation caused by harmful agents which may be found in the sample or opportunistic, invasive agents by extracting lipids from the sample.
It is another object to provide a method for lipid extraction having particular utility for storing tissue samples for longer periods of time than previously possible, e.g., for a period of several weeks, at room temperature.
Another object of the present invention is to provide a previously unknown product having decreased phospholipid content compared to fresh, or untreated, tissue samples, with a longer shelf life than previously possible.
These and other objects, as well as the several advantages of the invention, will be apparent to those skilled in the art upon reading the specification, the examples and the appended claims.
In accordance with the present invention, there is provided a method, or treatment, for extracting lipids from a collagenous tissue sample comprising immersing the sample in a high osmolality aqueous solution of a salt and a sugar, the salt being a salt selected from the group of salts which are capable of penetrating the sample, the sugar functioning to maintain the high osmolality of the solution even as salt concentration in the solution decreases as the salt penetrates the tissue sample, to decrease the bioburden of the sample as compared to the bioburden of untreated tissue samples. In a preferred embodiment, the salt and sugar are utilized in proportions in which the salt is utilized in a weight to volume ratio of higher than about 11.7% and the sugar is utilized in a concentration of about 30 to about 80% (W/V).
Also provided is a tissue sample having improved shelf life when maintained in an aqueous solution which is produced by immersing the tissue sample in a storage medium having an osmolality higher than about 4500 mosm, the storage medium comprising water, sucrose, and a halide salt, wherein the tissue sample is characterized by a decreased bioburden and a decreased lipid content relative to tissue samples which are not produced in this manner. In a preferred embodiment, the storage medium comprises about 30 to about 80% (W/V) sucrose, and the halide salt is utilized in a concentration of higher than about 11.7% (W/V).
Once removed from the animal, collagenous tissue can become hydrated and thicken. The thickening of the tissue is believed to be the result of the partial unwinding of the collagen fibrils, which makes the fibrils more susceptible to enzymatic degradation. The interaction of native helical collagen molecules inside the collagen fibrils must be kept intact in order to maintain the stability of the fibrils. To do so, it has been discovered that the ionic strength of the solution in which the sample is stored must be increased to such a point that the hydrophobic interaction between collagen molecules is maximized. This is accomplished by the use of a high salt concentration in the media solution of the present invention.
However, as the salt in the solution penetrates the tissue sample, and depending upon diffusion time across the thickness of the tissue and the actual thickness of the tissue, it is believed that a concentration gradient is set up between tissue and solution. As the salt concentration in the tissue increases to about one molar at physiological pH, the ionic interaction between collagen molecules in the fibril is interrupted by interaction between the collagen molecules and the salt, resulting in the unwinding of the fibril and subsequent solubilization of the fibril. It is therefore believed that the high concentration of sugar in the media solution of the present invention maintains the osmolality of the solution and serves as a component to cause the short term aggregation of the collagen fibrils. The osmolality (represented by the Greek letter xcexc) of the storage medium of the present invention is preferably higher than the osmolality of 3.0 M NaCl solution, e.g., 4500 mosm, and in a presently preferred embodiment, the osmolality is higher than about 6000 mosm. The upper limit of the osmolality is imposed by the practicalities of handling the solution, e.g., the increasing viscosity that results from high solute content. Of course, the osmolality is also limited by the ability of the solvent to hold solute, e.g., the point at which it is saturated. Both sugar and salt contribute to the osmolality of the medium of the present invention (as compared to the ionic strength of the solution, which results from inclusion of the salt, which dissociates in water) and the relative contributions of salt and sugar to the osmolality of the solution are not as important to the function of the solution as the total osmolality. Maintenance of the high osmolality of the solution in the face of depletion of the concentration of the salt by penetration into the tissue appears to mitigate collagen denaturation by maintaining the hydrophobic interaction of the collagen fibrils in the same manner as the salt functions to maintain collagen aggregation.
Many salts are suitable for use in the storage medium of the present invention, but those which will function as described above to penetrate a tissue sample to inhibit hydration of the proteinaceous material in its native configuration are specifically contemplated. Due to their low cost, high solubility, and ready availability, sodium chloride and potassium chloride are the preferred salts for use as the salt in the storage medium of the present invention, and consequently, the examples set out below refer to those salts. However, many other such salts are readily known in the art and readily available, including, for instance, the ammonium, sodium, calcium, magnesium, manganese, and potassium salts of halides, nitrites, nitrates, phosphites, phosphates, sulfites, sulfates, and alkanoic acids such as propionates, acetates, and formates, and specifically, the aforementioned sodium and potassium chloride, magnesium chloride, and sodium nitrite. All such salts, as well as the many which are not listed here but which would be recognized by those skilled in the art who have the benefit of this disclosure to function in substantially the same way to achieve substantially the same result as those which are listed, are contemplated by the present invention.
As a general guideline, the desired contribution to the osmolality of the storage medium of the present invention attributable to the salt component is achieved by inclusion of, in the case of NaCl, about 2.0 to about 5.0 M NaCl in the medium (e.g., from about 116.8 to about 284.0 g per kilogram (e.g., one liter) final volume of water, or about 11.7 to about 28.4% (W:V) concentration), the preferred range being about 2.25-4.0 M. For salts such as calcium chloride, the desired contribution to the osmolality of the medium is achieved by inclusion of from about 1.3 to about 3.4 M CaCl2 in the medium, and so on.
Similarly, many sugars can be used as the second component of the storage medium of the present invention. Again because of its low cost, high solubility, and ready availability, most of the examples set out below refer to the use of sucrose as the sugar in the storage medium. However, those skilled in the art will recognize that other sugars such as glucose, fructose, mannose, galactose and any other monosaccharides, disaccharides such as maltose, cellobiose, and lactose, trisaccharides, or polysaccharides such as amylose or amylopectin, as well as sugar derivatives such as sorbitol (derived from glucose by reduction of the aldehyde group), mannitol, etc., glycosides such as methyl glucoside (derived from glucose by acid-catalyzed reaction of methanol with glucose), or proteoglycans will also function in substantially the same way to achieve the desired result of maintaining the proteineous materials of a tissue sample in their native state, and all such sugars are contemplated as falling within the scope of the present invention.
In the case of sucrose, the desired contribution to the osmolality of the storage medium of the present invention can generally be obtained by using a concentration of from about 30 to about 85% W:V (e.g., from about 0.3 to about 0.85 kg per kg, e.g., one liter, final volume of water) sucrose, the preferred range being from about 30 to about 80% W:V concentration, e.g., about 0.85 to about 2.35 M sucrose. In other words, the contribution of the sugar to the total osmolality of the storage medium of the present invention ranges from about 3400 mosm upwardly to the saturation point of the solvent.
In some salts which are not as soluble as others, the desired contribution to the osmolality of the media is obtained by increasing the amount of sugar in the solution. By compensating for solubility in this manner, satisfactory results are obtained even with certain salts such as potassium chloride, potassium iodide, sodium citrate, sodium acetate, and sodium sulfate. These salts are listed here because it was discovered that, at least when the media of the present invention is made by the following method, they were not completely solublized. This method, which is but one way to prepare the media and is described here to better illustrate the teaching set out above as to the upper limits on the concentration of the salt imposed by the practicalities of dissolving one or both of the components of the solution, was an attempt to prepare the solution of the present invention using various salts and a standard of 67 g of sucrose per 100 ml final volume. Because sugar occupies a large volume, the first step was to attempt to dissolve 0.3 moles of various salts in 40 ml PBS by overnight stirring at room temperature. If soluble, then the 67 g sucrose was added (again by stirring overnight at room temperature) and the final volume adjusted 100 ml with PBS to prepare the 3 M salt and 67% W:V sucrose solution that was desired. The combinations of salts and sucrose were as follows:
The weights of each salt added were calculated for a total of 0.3 moles of salt to be added for each solution and take into account the total molecular weight of the salt as provided in the anhydrous or hydrated form (N/A=not applicable).
In a second set of experiments, the media for use in accordance with the improved method of the present invention was prepared using a standard of 17.5 g sodium chloride along with 67 g of various sugars per 100 ml final volume. Because the sugar occupies a large portion of the volume, the first step was to dissolve the 17.5 g of sodium chloride in 40 ml PBS then 67 g of the particular sugar was added. The final volume was adjusted to 100 ml with PBS to prepare the 3 M salt and 67% W:V sucrose solution that was desired.
The fructose was not completely soluble in the total volume of 100 ml. Therefore, an additional 33 ml PBS was added to solublize all of the components. Thus, this solution was 50% fructose and 2.25 M NaCl. It will be understood by those skilled in the art who have the benefit of this disclosure that those solutions which were not able to be prepared by these methods may be prepared by other methods known in the art and that even a solution such as the sodium chloride-fructose solution, in which additional PBS was added to solublize the fructose, gave satisfactory results (as set out below) when used in accordance with the present invention.
Although the examples set out above disclose a number of high osmolality solutions which are used to advantage in the method of the present invention, there are many other such solutions which may also be utilized to advantage, including solutions which are comprised of the very salts and/or sugars listed above as being insoluble by the method described in those examples. Those skilled in the art who have the benefit of this disclosure will recognize that additional solute can be solublized by using, for instance, elevated temperature, more efficient and more vigorous stirring and/or agitation, and by other methods known in the art.
It will also be recognized that some salts cause a decrease in pH when dissolved (note the pH of the above-described solutions including manganese sulfate and magnesium chloride). Further, this lowering of pH can have detrimental effect(s) on the collagenous tissue to be cross-linked. Reference is made, for instance, to Example 4, infra, wherein it is reported that a solution made in accordance with the present invention and comprised of magnesium chloride and sucrose had a pH of 3.59. When that tissue was evaluated by heat shrink test, there was no sharp decrease in tissue length as a function of temperature, indicating that the tissue was likely damaged by this low pH. Even so, the magnesium chloride/sucrose solution can be used to advantage in connection with the present invention by, for instance, neutralizing the acidity of the solution by addition of sufficient magnesium hydroxide to raise the pH or by using a stronger butter. Such adjustments in pH are known to those skilled in the art and the resulting solution gives satisfactory results when used in the method of the present invention.
The storage medium is buffered to physiological pH by use of any of a number of commonly used buffers, one of the most commonly available being phosphate buffered saline (PBS), and that buffer is used to advantage in the medium of the present invention. Other suitable buffers include those containing potassium or sodium phosphate, or potassium or sodium chloride, such as a Good""s buffer, e.g., HEPES, TES, or BES (Research Organics, Inc.), preferably at concentrations of from about 0.2 to about 1.0 M. However, the molar concentration of the buffer is not as important as the concentration of the other two components of the storage solution of the present invention. Almost any concentration of buffer components which will maintain pH between about 3.5 and about 10, and preferably at about physiological pH, will function effectively in connection with the storage medium of the present invention. By reference to the term xe2x80x9cphysiological pHxe2x80x9d herein, it is intended to refer to nominal hydrogen ion concentration in vivo; those skilled in the art will recognize, and the term is specifically intended to encompass, that a pH range of from about 6.8 to about 8.6 may be encountered, depending upon the system, in normal living systems.
The following non-limiting examples describe the invention in further detail.